WO2002069702A1 - Procede de conservation d'un organe de mammifere - Google Patents

Procede de conservation d'un organe de mammifere Download PDF

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Publication number
WO2002069702A1
WO2002069702A1 PCT/JP2002/002074 JP0202074W WO02069702A1 WO 2002069702 A1 WO2002069702 A1 WO 2002069702A1 JP 0202074 W JP0202074 W JP 0202074W WO 02069702 A1 WO02069702 A1 WO 02069702A1
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Prior art keywords
heart
organ
water
gas
dehydration
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PCT/JP2002/002074
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English (en)
Japanese (ja)
Inventor
Kunihiro Seki
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Biobank Co., Ltd.
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Application filed by Biobank Co., Ltd. filed Critical Biobank Co., Ltd.
Priority to EP02702761A priority Critical patent/EP1380207A4/fr
Priority to JP2002568896A priority patent/JPWO2002069702A1/ja
Priority to KR10-2003-7011572A priority patent/KR20040008131A/ko
Publication of WO2002069702A1 publication Critical patent/WO2002069702A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids

Definitions

  • the present invention relates to a technique for preserving mammalian organs in vitro for a long period of time.
  • cryopreservation is the mainstream, and the effective storage time for organ transplantation is about 4 to 24 hours.
  • trehalose (CH ⁇ Ou) is a non-reducing disaccharide widely distributed in nature and has been reported to have a stabilizing or protecting effect on the cell membrane structure under various stresses (Crowe JH). , Crowe LM, Chapman D; Science 233, 701-703, 1984 and Wiemken A; Antinei Van Leeunwenhoek. 58, 209-217, 1990).
  • Trehalose has been reported to protect cell membranes when the heart is exposed to 4 ° C low-temperature ischemic injury (Stringham JC, Southhard JH, Hegge J, et all; Transplantation, 58, 287-294, 1992 and Hirata T, Fukuse T, Liu CJ, et all; Surgery, 115, 102-107, 1994). It has been reported that trehalose increases by 10 times in anhydrous state in a high hydrostatic pressure experiment of a viper (Crowe JH, Crowe LM, Chapman D; Science 233, 701-703, 1984 and Crowe JH). , Crowe LM, Chapman D, Aurell Wistorm C; Biochemical Journal, 242, 1-10, 1987). This beetle is a multicellular organism composed of about 40,000 cells and also having nerve cells.
  • the present inventor has discovered that dry beetles and the like can withstand the load of a high water pressure environment as extreme as 600 MPa in perfluorocarbon solution and retain vitality (Kunihiro Seki et al; Nature Vol. .395, No. 6705, pp. 853-854, 29 Oct. 1998, and JP-A-11-289917, the contents of which are incorporated herein by reference).
  • the dry state of the viper is formed by the tan state or the barrel state. Its physiological mechanism is not fully understood, but the viper is dehydrated due to an extremely low water content in the body.
  • the metabolic function of the organ is lowered by lowering the temperature of the organ, and the organ is kept alive.
  • the metabolic function is reduced by lowering the temperature, abundant ions are present in the polar medium, water, which causes cell self-destruction, cell death, and necrosis over time.
  • refrigerated storage has the unavoidable possibility that severe thrombosis and dysfunction rapidly increase as the storage time is prolonged, and there is a qualitative limit to the storage period.
  • Organs preserved by the method disclosed in JP-A-2000-72601 can be used to collect living nerves and stem cells after resuscitation, and the organs themselves or the collected tissues can be used for transplantation medicine.
  • organs themselves or the collected tissues can be used for transplantation medicine.
  • histopathological studies the long-term storage of biomaterials that can resuscitate rather than necrotic specimens is significant.
  • the above-mentioned storage method can prevent the self-destruction of cells and tissues of an organ stored outside a living body over time and greatly increase the number of storage days.
  • the dehydration method depends on a dehydrating agent that comes into contact with the organ. ing. In order to further improve its storage method, it is necessary to search for a better dehydration method. Disclosure of the invention
  • the present invention provides a method for removing an organ such as a mammalian heart, comprising: a dehydration step of removing water from the organ and leaving a revivable amount of water; and a step of immersing the organ in an inert medium and maintaining the organ at a refrigerated temperature. Regarding how to save.
  • the method for preserving mammalian organs comprises the steps of: extracting water from an organ containing a water content that is physiologically common sense through the vascular system to a weight ratio of about 10 to the total weight of the organ before dehydration.
  • a dehydration step of removing at least about 25% or more of water and leaving about 10 to about 20% or more of water by weight with respect to the total amount of water before dehydration, and immersing the organ in an inert medium. And maintaining the temperature at the refrigeration temperature.
  • the above organs include heart, liver, kidney, And lungs.
  • the method for preserving the heart of a mammal includes a blood removal step of perfusing a physiological saline solution into the heart to replace blood in the heart with a physiological saline solution; A dehydration step of deriving water in the organ to remove about 10 to about 50% by weight of water relative to the total weight of the heart before dehydration, and immersing the heart in an inert medium for about 1 to about 8 ° Maintaining the temperature at the refrigeration temperature of C.
  • the invention may also theoretically include a dehydration step to remove from about 25% to about 60% water by weight relative to the total weight of the heart before dehydration.
  • Dehydration using the vascular system involves delivering gas to the vascular system of an organ or heart.
  • the dehydration step may further include contacting the organ, which is immersed in an inert medium, with a dehydrating agent.
  • a dehydrating agent it is preferable to remove blood from the organ using a physiological salt solution prior to the dehydration step in order to avoid the problem of blood coagulating by contact with the perfused gas.
  • blood removal perfusion where the flow pressure is constant.
  • the gas fed into the vascular system is preferably air, an o 2 —co 2 mixture, but an inert gas such as N 2 , He, Ar, Ne, Kr or Xe is used. May be.
  • an inert gas such as N 2 , He, Ar, Ne, Kr or Xe is used. May be.
  • a perfusion device for perfusing a physiological salt solution and to feed gas instead of the physiological salt solution.
  • liquid to be introduced into the vascular system examples include a solvent that uses an osmotic pressure difference to move water from cells, such as a hypertonic solution having a higher concentration than the body fluid of an organ. Also, an inert medium described later or alcohols may be used.
  • the fluid enters the capillary of the vascular system to generate a fluid pressure, reaches almost all of the organ tissues, and circulates through the capillary (eg, from an arterial blood vessel to a venous blood vessel) to reach the other end of the vascular system. .
  • This dehydration uses the water supply pathway that is unique to the organ, so that individual tissues and cells can be targeted to create a loose and uniform dehydration state.
  • Even multi-cellular and multi-tissue organs of mammals can smoothly transition to a highly dehydrated state without applying unnecessary stress to living tissues. Therefore, usually 10 wt.
  • Even with dehydration of 25 wt% or more, fl organs can reach an extremely stable asphyxia state without ischemic injury or damage to living tissues. Then, when water is regained, the function is restored, and the function of not only the cells and tissues but also the organ itself can be revived.
  • the asphyxia state means a biological asphyxia, but the asphyxia intended in the present invention means that the appearance of life phenomenon is not recognized due to the high dehydration state, but the life phenomenon is resumed when the water is recovered. Means a state that can be recognized.
  • the term “resuscitation” as used in the present invention means a phenomenon in which, when water is regained from a dehydrated state, an electrophysiological reaction of a tissue or biological biological activity as an organ is recognized.
  • the present invention provides a step of forming an oil film on the surface of an organ containing a physiologically common water content, and exposing the heart to a gas to evaporate water in the heart into the gas. Removing more than about 10% by weight of water relative to the total weight of the heart before dehydration, and maintaining the organ at a refrigerated temperature of about 1 to about 8 C, preferably about 2 to about 4. And a method for preserving the heart of a mammal.
  • the present invention also provides a blood removal step of perfusing a physiological saline solution into the heart to replace blood in the heart with a physiological saline solution, a step of forming an oil film on the surface of the removed blood heart, Exposing water to a gas to evaporate the water in the heart into the gas to remove about 10 to about 50% of water by weight relative to the total weight of the heart before dehydration; Maintaining the refrigeration temperature of about 8 ° C to about 8 ° C.
  • biological structures such as biological membranes are less susceptible to attack by substances that can be activated in the aqueous phase, especially metal ions.
  • the biological structure is considered to be protected by being surrounded by crystalline water called bound water.
  • bound water As a result of the removal of the polar medium that degrades living tissues, tissues and cells can escape irreversible decay over time, and qualitative improvements in the preservation of organs can be achieved.
  • trehalose in the preservation solution can contribute to stabilization of the biological structure.
  • the heart, liver, kidney, knee, or lung preserved by the method of the present invention may contain a body fluid warmed to about body temperature or a substitute body fluid, such as the above physiological salt solution, artificial blood and / or blood, in the vascular system.
  • the resuscitated organ could be transplanted into the human body with the organ or tissue taken from the organ. Nervous tissue and other living tissues and cells can be collected from resuscitated organs and used for pharmacological tests.
  • the present invention can be applied to organs of mammals other than humans for the purpose of xenotransplantation into the human body, and thereby provides a technology for preserving animal organs, for which clinical demand is expected to increase due to lack of donors.
  • the present invention is useful for preserving organs that can be mass-produced, such as hearts, livers, and kidneys in cultured fish, and uses a preservation technique that can withstand long-term transport, especially air transport that takes more than ten hours. Will provide.
  • technologies that enable long-term preservation such as the present invention, will help establish an organ bank. For example, it is possible to regenerate desired tissues and organs by culturing totipotent stem cells such as embryonic stem cells, and to apply the present invention to preserving them. Preserving spare organs before the disease is discovered will allow them to receive a transplant immediately after the onset of the disease. Further, the present invention may be useful for preserving the brain and other nervous tissues.
  • the “removal of about 10% or more of water” or “removal of about 25% or more of water” in the present invention is intended to remove body fluids in the vascular system and individual cells and free water between cells.
  • the expression "leave about 10 to about 20% or more of water” as used in the present invention means that the living tissue contains bound water and has a sufficient amount of water for resuscitation.
  • it is considered that what determines the freshness of the preserved organ is mainly the amount of free water, and it is theoretically preferable to remove free water as much as possible in order to prolong the storage time.
  • Bound water is water that can be observed in a hydrated or crystalline state, and free water can be defined as water other than bound water.
  • the vascular system is anatomically classified into the vascular and lymphatic systems.
  • the term "vascular system" as used in the present invention preferably includes a nutritional vasculature for supplying water and nutrients to individual cells of an organ.
  • a perfusion device can be connected to a unique vessel connecting to the atria and ventricles to indirectly apply fluid pressure to the coronary arteries and coronary veins connected to the nutritional vasculature of the heart.
  • a functional vasculature may be used in an organ such as the liver.
  • organ containing a water content that is physiologically common sense in the present invention is typically an organ that has been removed from a living body. It can be understood that the blood is replaced with a physiological salt solution by perfusion for the organ. The amount of dehydration can be specified based on the weight of an organ having such a physiologically normal water content.
  • the “physiological saline solution” referred to in the present invention is a body fluid substitute having the same biological activity as blood, and is typically a known Ringer's solution, for example, a KH (Kreps-Henseleit) solution.
  • a physiological salt solution may be used by dissolving a polysaccharide which will help to stabilize a dehydrated anatomy.
  • polysaccharides are preferably trehalose.
  • amino acids such as malic acid, mannitol, glycerol, glycine benzoin, puline, and ectoin can be used by dissolving them in a physiological salt solution.
  • the "inert medium” referred to in the present invention is a medium that is insoluble in water and oil, and is preferably a fluorcarbon solution which is liquid at the storage temperature, particularly a perfluorocarbon solution. Further, as long as similar conditions are satisfied, not only liquid but also gas, sol or gel may be used. Mercury and silicone oil may be used as other inert media.
  • FIG. 1 is an electrocardiogram recorded in Experiment 8 of Example 3.
  • FIG. 2 is an electrocardiogram recorded in Experiment 9 of Example 3.
  • FIG. 3 is an electrocardiogram recorded in Experiment 1 of Example 4.
  • FIG. 4 is an electrocardiogram recorded in Experiment 2 of Example 4.
  • FIG. 5 is an electrocardiogram recorded in the experiment of Example 5.
  • the organ to be preserved for example, the organ removed by surgery, is washed and bled.
  • a perfusion apparatus for blood removal a known Langendorf f apparatus can be used. The technique of blood removal perfusion is based on the Langendorf f method (Doring H. J, Dehnert H; Biomess tec nik-Ver tag Match Gmb, Germany). , 1988).
  • an organ is attached to the aorta of the vascular system with a perfusion catheter (or force generator), and a trehalose-KH (Kreps-Henseleit) mixture is injected into the vascular system.
  • a perfusion catheter or force generator
  • a trehalose-KH Kerr-Henseleit
  • the physiological salt solution is preliminarily subjected to oxygen aeration (aeration), so that a fresh substitute body fluid is always sent into the organ.
  • oxygen aeration oxygen aeration
  • the temperature of the organ is reduced to 1 to 8 ° C, and the activity of the organ is stopped. Enter the dehydration process following blood removal.
  • the dehydration step is achieved by using the Langendor ff device used for the blood removal.
  • gas is sent from the perfusion device to the vena cava of an organ after blood removal is completed at a predetermined flow pressure instead of a physiological saline solution.
  • the artificial perfusion means is not limited to a means for unilaterally sending gas from the aorta, but includes a closed circulatory system connected between the aorta and the vena cava. That is, the gas perfusion is not limited to a method of feeding a gas, but may be a method of suctioning a vascular system.
  • dehydration proceeds so that the physiological saline solution flows out from the vena cava in the organ.
  • the gas that has entered the blood vessel flows through the individual capillaries to the vena cava side, and the individual cells and intercellular water flow out into the gas flowing through the blood vessel due to their vapor pressure.
  • most of the free water in each tissue and cell is drained out of the organs through the vasculature.
  • the above-mentioned dehydration using the vascular system unlike the treatment in which a dehydrating agent is brought into contact, utilizes the extremely large surface area of the capillaries that are uniformly distributed in the organ, and allows the tissue to grow slowly but in a relatively short time. And individual cells can be dehydrated to a target.
  • This advantage is apparent from the fact that there is less dehydration unevenness compared to the above-mentioned dehydrating agent and the degree of discoloration of the dehydrated organ is not serious.
  • dehydration by gas perfusion actually resulted in a very high resuscitation rate and improved resuscitation status.
  • gas dehydration is an active process that can be controlled from the outside, and is a less stressful and efficient means for organs that require quick and careful handling.
  • the gas delivered to the vascular system may be inexpensive compressed gas packed in commercial cylinders. o 2 - If C 0 2 mixed gas, preferably those containing C_ ⁇ 2 of less than 5%. In the embodiments described later, a dry gas is used, but a gas containing moisture may be used. Furthermore as a gas for dehydration will more preferably may use a N 2, H e, A r , inert gases such as N e, K r or X e. Among the inert gases, Xe is expensive, but the anesthesia of living tissue has been reported, so that the advantage of using Xe for the dehydration of the present invention is expected to be great. The use of an inert gas also has the purpose of avoiding damage to living tissues due to active oxygen.
  • a dehydration method in which a dehydrating agent is brought into direct or indirect contact with an organ during storage can also be used together.
  • a simple method of immersing the dehydrating agent adjusted to a desired volume together with the organ in an inert medium described later is simple.
  • an organ surrounded by silica gel, molecular sieve, zeolite, or the like is housed in a wire mesh, a synthetic fiber net, or the like, which is a material inert to an inert storage medium. Immerse in an inert medium.
  • the amount of the dehydrating agent to be used can be calculated from the water removal rate required for the organ as described below, and the dehydrating agent may be removed, added or replaced at an appropriate timing after immersion.
  • the organ immediately before dehydration contains a substitute fluid and contains physiologically common water content. For long-term storage, remove as much free water as possible from the water content.
  • the amount of water to be removed is defined by a water removal rate (weight ratio) based on the total weight of the organ represented by the following formula.
  • Water removal rate (weight ratio) 100— (total weight of organ after dehydration ⁇ total weight of organ before dehydration x 100)
  • the absolute amount of water molecules in the inside or the state thereof may be specified.
  • NMR is the most widely used technique for studying the state of water in living tissues.
  • water consists of three states: about 8% of water molecules are bound water in which biological molecules such as the inner membrane of cell membranes and proteins and nucleic acids are bound, and about 82 water molecules are: In the free water other than the bound water, the remaining 10% of the water molecules are free water in contact with the outside of the cell membrane.
  • the bound water unlike other water molecules, maintains a certain predominant arrangement and is oriented over several molecular layers based on the water molecules directly bonded to the biopolymer.
  • bound water accounts for about 10 to about 20% and free water for about 80 to about 90% of the total water content in living tissue.
  • the typical total water content is about 80% by mass, based on the total organ weight, and the bound water is 8-16% ⁇ free water is 64-72% by mass.
  • the mass of free water remaining in the rat heart can be reduced to several percent to 40%, but the absolute amount of bound water does not change. Even if the actual water content varies slightly depending on the animal species and organ type, the removal of about 25% or more of the free water by weight relative to the total weight of the organ before dehydration will result in a weight ratio based on the total water content before dehydration. About 10 to 20% or more of water (including bound water) can be left.
  • the percentage of free water allowed for an organ depends on the method of dehydration, type of organ, intended storage period, and other storage conditions. At present, approximately 25-35% is considered safe in rat hearts. For porcine heart, about 10-50%, especially 15-25%, may be appropriate.
  • Dehydrated organs are stored in perfluorocarbon, an inert medium that is insoluble in water and oil.
  • the immersion of the organ in the inert medium is generally performed in a sealed state under normal pressure, and may be performed in a pressurized state as needed. At this time, it may be stored together with the dehydrating agent as described above.
  • the inert medium is preferably aerated with pure oxygen.
  • the "temperature below the refrigeration temperature” in the present invention is about +1 to about + 8 ° C, preferably about +2 to about + 4 ° C, and a predetermined number of days is maintained in this storage state.
  • the free water of the organ is sufficiently removed, it is theoretically possible to store it at the freezing temperature, for example, in liquid nitrogen.
  • the above-mentioned Langendor ff method can be used for resuscitation from the preserved state of the organ.
  • the organ is placed in a KH solution in a petri dish at + 4 ° C, preferably in a KH solution that has been agitated with pure oxygen.
  • a perfusion catheter is fixed to the aorta of this organ, and the KH solution, which has been continuously aerated with an o 2 —co 2 gas mixture and warmed to 37 ° C., is supplied at a constant flow rate by a perfusion pump to the large artery. Feed into catheter. By this perfusion, resuscitation of the organ is achieved.
  • the problem with organ preservation in mammals is that it is necessary to verify which tissues of the organ survive after preservation. These include tissue anatomical methods, transplantation methods for actual transplantation and verification, and electrophysiological methods. Regardless of which approach is used, the first step is to verify whether the tissue cells are alive.
  • an electrophysiological technique for example, an electrocardiographic measurement on the heart was used.
  • the electrocardiogram can record the activity of the nerve cell tissue in real time, and can determine that cell death has occurred when the activity of the nerve cell disappears.
  • the resuscitation state of the organ can be observed by visually confirming the discoloration of the organ tissue and, in the case of the heart, the presence or absence of pulsation.
  • Invention involving formation of oil film and dehydration by exposure to gas
  • the present invention uses the evaporation of water by exposing organs to gas instead of the vascular dehydration described above.
  • the blood removal step and the regenerating step are basically the same as the above-mentioned invention by dehydration from the vascular system.
  • the preservation step may or may not employ the above-described preservation method of immersing an organ in an inert medium. If the organs are not immersed in an inert medium, the dried organs are kept at a refrigerated temperature in a gas of appropriate humidity for the desired storage period.
  • the removed organs are removed from the perfusion device together with the forcenula and immersed in oil.
  • An oil film is formed on the entire surface of the organ. The formation of an oil film can prevent unevenness in the drying rate of the isolated heart, which is threatened by the removal of water under dry gas, and consequent contraction and keratinization of the heart surface.
  • the oil used is preferably harmless to the organs and has a viscosity capable of forming an oil film of an appropriate thickness on the surface of the organs, and is typically silicone oil.
  • Silicon oil is composed of organic silicon oxide polymer and is almost physiologically harmless and chemically inert.
  • the silicone oil used in the examples described below is non-volatile with a kinematic viscosity of 100/2 / S (25 ° C) and is considered to be suitable for protecting the heart surface from overdrying for a long time. It can be visually confirmed that when an oil film is formed on the surface of the heart, physical damage such as cracks on the surface of the heart caused by drying in the absence of oil is reduced.
  • Gas used is typically air, or o 2 - co 2 but the mixed gas can be used, but are not limited to these.
  • the gas for drying ⁇ 2 - CO 2 mixed gas is preferred.
  • a gas containing Xe may be used as the gas for storage. Since xenon is an inert gas without polarity, it can be hydrophobically hydrated with surrounding water to suppress the thermal motion of water molecules. As the structuring of water increases and the molecular movement slows down, the viscosity of water increases, and biological reactions take place via water, so it is thought that increasing the viscosity of water suppresses cell metabolism.
  • xenon as a storage gas, in addition to the removal of free water and maintenance at low temperatures, further suppresses organ metabolism.
  • Xenon is known to have a dissociation pressure (0 ° C) of 1.15 atm (0.1115M Pa), which makes it easier to form gas hydrates at relatively low pressure. Therefore, when using xenon, the organs should be stored under pressure. In a pressurized environment, organs (cells It is thought that the solubility of xenon in the aqueous phase inside (including inside and outside) is increased. It is presumed that pressurization of xenon surrounding the organ promotes the structuring of water in the organ and significantly suppresses organ metabolism.
  • the dehydration method using gas exposure includes, for example, putting a degassed organ in a sealed container kept at refrigeration temperature with a silica gel desiccant, and drying the organ for a predetermined period. Exposure to exposed gas.
  • the dehydration rate to be achieved depends on the weight of the organ, but is 10-50% of the total organ weight before dehydration. However, under the same drying conditions, the achieved dehydration rate depends on the size (weight, etc.) of the organ, so it is considered preferable to apply drying conditions taking this into account.
  • the time for dehydration depends on the weight of the organ and the amount of desiccant. For example, a rat heart is about 24 to 48 hours, and a bush heart is about a week, but not limited thereto. After each of these drying periods, the organs may be stored in an inert medium.
  • a high dehydration rate desirable for long-term storage can be achieved without damaging the organ. Due to less damage to the organs, resuscitation is very good during relatively short-term storage, and the resuscitation rate increases with long-term storage.
  • Example 4 the pig organ was successfully revived after 37 days. It was confirmed that the myocardial tissue of the organ was revived during the period in which the organ decayed by conventional refrigerated storage.
  • Example 1 Storage of rat heart dehydrated via vascular system
  • Rats were intraperitoneally injected with 0.25 ml of neptal and 5 mg of heparin sodium (Heparin Sodium Salt), and weighed before starting heart extraction.
  • the excised heart insert the catheter into the aorta placed in a constant temperature bath, dissolving the trehalose 117 thigh ol 95 ⁇ 2 - 5% C_ ⁇ 2 gas mixture with the KH solution aerated, Langendorf f formula Perfusion was performed. The temperature of the thermostat was lowered to stop the heart, and the blood removal and perfusion was completed.
  • the perfusion apparatus After measuring the weight of the heart, the perfusion apparatus sent the above 95% O 2 — 5 C 5 2 mixed gas to the heart to start dehydration. Start dehydration while weighing the heart and recording the water removal rate One hour and 30 minutes after, the gas perfusion was stopped. The total weight of the heart was measured with the outer surface simply wiped off moisture.
  • the dehydrated heart is immersed in 500 ml of 4 perfluorocarbon solution (Florinato FC77; C8 F17, manufactured by Sumitomo 3LM Co., Ltd.) which was previously agitated with pure oxygen for 1 minute, and placed in a sealed container. And stored in the refrigerator.
  • 4 perfluorocarbon solution Fluorocarbon solution
  • the preserved heart was removed from the perfluorocarbon solution, a catheter was introduced into the aorta, and resuscitation was performed by 1 angendor ff perfusion using a warm KH solution in which the above gas mixture was violated. .
  • Example 2 Storage of rat heart dehydrated via vascular system
  • a solution of 5 mg of heparin sodium dissolved in 0.3 ml of physiological saline was intraperitoneally administered.
  • a catheter was inserted into the aorta of the removed heart and ligated with a cotton thread.
  • the isolated heart into which the catheter had been inserted was attached to a constant flow perfusion apparatus, and a KH solution at 27 ° C in which trehalose was dissolved in 117% was perfused at 3.2 ml / min.
  • the KH solution was constantly aerated with a gas mixture of 95% 0 2 — 53 ⁇ 4 C ⁇ 2 .
  • the heart was dried by perfusing air gas at a flow pressure of 0.1 kgf / cm 2 from one ter. After measuring the weight of the dried heart, it was immersed and stored in PFC kept at 4 ° C while constantly aerating with a mixed gas. Four hours later, the heart was taken out of the preservation solution, attached to a constant flow perfusion apparatus, and perfused with a KH solution at 27 ° C constantly aerated with a mixed gas at 3.2 ml / min. It is known that, when the isolated heart starts perfusion again after storage, if the tissue cells are still alive, they will be resuscitated autonomously and neural activity will appear. Electrodes were attached to the resuscitated heart, and surface electrocardiograms were recorded with a pen oscillograph.
  • the experiment was performed in the same manner as in Experimental Example 12 except that the storage time was changed to 16 hours.
  • silicone oil was applied to an isolated heart from a rat, and the heart was stored at 4 ° C. for 3 days under the pressure of dry gas.
  • the rats used in this experiment were 7-week-old male Wistar strains (300 g) that had been artificially bred in accordance with the NIH laboratory animal standards in the United States.
  • the heart was extirpated from the rat, and the heart was bled. After that, 2 ml of a cardiomyopathy (Mi 0 teeter Mochida Pharmaceutical) was injected through a catheter to stop the heart.
  • a cardiomyopathy Mi 0 teeter Mochida Pharmaceutical
  • the surface of the heart was coated with silicone oil (WF-30 Wako Pure Chemical), wrapped in sterile gauze (120 X I 20 mm), and placed in a standard bottle (60 ml).
  • the bottle containing the heart was placed in a pressure-resistant chamber lined with silica gel and sealed.
  • a mixed gas (95% oxygen, 5% carbon dioxide)
  • the mixed gas was sealed to 0.2 MPa.
  • the chamber was stored in a refrigerator at 4 degrees Celsius.
  • the chamber was opened every 24 hours and silicone oil was reapplied to the isolated heart.
  • the heart was removed from the silicon oil in the chamber and set in a constant-pressure perfusion apparatus.
  • the isolated heart started perfusion at 37 degrees Celsius.
  • ECG recording electrodes were attached to the left ventricle and the aortic orifice, and electrocardiograms were continuously recorded by hyperbolic lead using a biological amplifier (Bioview-ENEC Sanei).
  • cardioplegic solution cardioprotective solution
  • the gas sealed in the chamber was changed, and the drying period was changed. After the drying period, the sample was immersed in a preservative solution (silicone oil) and stored under pressure in a chamber as in the case of drying.
  • a preservative solution silicone oil
  • Heart weight before starting resuscitation 0.89 g (35% moisture removal)
  • Heart weight before storage 1.37g
  • FIG. 1 is a heart surface electrocardiogram in this experiment (21:04 measurement). Heart weight before storage: 1.61g
  • Figure 2 shows the heart surface electrocardiogram in this experiment (20:25 measurement)
  • the heart was exposed to a gas mixture to dehydrate and stored under the gas mixture for 7-8 days.
  • the oil-coated heart was wrapped in polyurethane foam and placed in a container (bottle), and the entire container was placed in a chamber and replaced with a mixed gas. It was kept in a refrigerator at a refrigerated temperature of 2-4 ° C. The organs were removed daily and reapplied with silicone oil.
  • FIG. 3 is an electrocardiogram of the surface of the heart resuscitated after 7 days of storage.
  • the oil-coated heart was wrapped in polyurethane foam and placed in a container (bottle), and the entire container was placed in a chamber and replaced with a mixed gas. It was kept in a refrigerator at a refrigerated temperature of 2-4 ° C. The organs were removed daily and reapplied with silicone oil.
  • FIG. 4 is an electrocardiogram of the heart surface resuscitated after 8 days of storage.
  • Example 5 Long-term storage of pig heart dehydrated by exposure to gas
  • long-term storage (37 days) was attempted by applying dehydration by exposing a pig heart to gas.
  • the organs were removed 37 days after the start of dehydration and perfused for resuscitation.

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Abstract

L'invention concerne un procédé de conservation d'un organe de mammifère, ce procédé comprenant une étape de déshydratation au cours de laquelle environ au moins 10 % en poids, de préférence au moins 25 % en poids, d'humidité est éliminée de cet organe, lequel contient une quantité physiologiquement acceptable d'humidité, par attraction de l'humidité dans l'organe par l'intermédiaire du système vasculaire, ainsi qu'une étape de trempage de cet organe dans un milieu inerte et de maintien de l'organe à une température de réfrigération.
PCT/JP2002/002074 2001-03-06 2002-03-06 Procede de conservation d'un organe de mammifere WO2002069702A1 (fr)

Priority Applications (3)

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EP02702761A EP1380207A4 (fr) 2001-03-06 2002-03-06 Procede de conservation d'un organe de mammifere
JP2002568896A JPWO2002069702A1 (ja) 2001-03-06 2002-03-06 哺乳類動物の臓器保存方法
KR10-2003-7011572A KR20040008131A (ko) 2001-03-06 2002-03-06 포유류 동물의 장기 보존방법

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US09/799,112 US6475716B1 (en) 2001-03-06 2001-03-06 Method for preserving mammalian organs
US09/799,112 2001-03-06

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CN102960335A (zh) * 2012-10-22 2013-03-13 同济大学附属上海市肺科医院 应用于无心跳供体肺移植中肺脏低温、通气序贯保存的装置
CN102960335B (zh) * 2012-10-22 2013-12-25 同济大学附属上海市肺科医院 应用于无心跳供体肺移植中肺脏低温、通气序贯保存的装置

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RU2003129508A (ru) 2005-04-10
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CN1505474A (zh) 2004-06-16
US6475716B1 (en) 2002-11-05
US20030022148A1 (en) 2003-01-30
EP1380207A4 (fr) 2004-11-03
KR20040008131A (ko) 2004-01-28

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